Previous evidence from our laboratory suggested that the products of the phage T4 DNA-delay genes 39, 52 and 60 either modify the E. coli DNA gyrase or specify a separate gyrase. We are now proposing to test the expectation that DD mutations alter the degree of supercoiling of the phage DNA replicative intermediate, using the techniques of ethidium bromide sucrose gradient sedimentation and electron microscopy. Our earlier work also indicated that the product of DD gene 52 is necessary for initiating DNA replicative forks. We are now planning to determine if gene 39 and 60 functions are also required for initiating replication. The experimental procedure involves estimating the frequency of growing points per unit of replicating DNA from measurements of DNA elongation rate (using autoradiography) and overall DNA increase. In further work, we plan to investigate the relationship of the host membrane to the DD gene products by altering the lipid composition of the membrane in DD am mutant infections. In these experiments a specific inhibitor of unsaturated fatty acid synthesis and E. coli mutants defective in this pathway will be employed. In related work, the role of the gyrase in recombination and mutation will be investigated, using DD am mutants. We have also observed that intracellular DNA in DD mutant infections appears to spread more readily than wild-type DNA in autoradiographic preparations. We now plan to use this property to try to visualize intact replicative intermediates. In the area of DNA repair, we plan to study the gene functions required for the recombinational repair of psoralen crosslinks in DNA, and the error-prone repair of psoralen monoadducts. These studies will employ phage and host mutants defective in DNA repair, and a procedure involving DNA denaturation-renaturation to measure removal of crosslinks. In addition, we would like to investigate whether the removal of the majority of EMS-induced DNA lesions which are neither lethal nor mutagenic, but which may cause incremental losses of function, are subject to recombinational repair.